Serum cholesterol lowering agent

ABSTRACT

A method of lowering serum cholesterol levels comprising administering to a patient in need of such treatment an effective amount of Z-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol, as well as pharmaceutical compositions useful in the method, is disclosed.

This is a 371 PCT/FI97/00140 filed Mar. 4, 1997

This invention relates to the use ofZ-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol for loweringserum total or LDL cholesterol or increasing serum HDL cholesterol.

It has been demonstrated that elevated levels of serum cholesterolassociated with low density lipoproteins (LDL) are a major contributingfactor in the development and progression of atherosclerosis. Low serumHDL cholesterol is an independent risk factor for atherosclerosis.Therefore it is desirable to provide a method for reducing serum totalcholesterol or LDL cholesterol levels combined with an increasing effecton HDL cholesterol levels in patients with hypercholesterolemia or atrisk of developing hypercholesterolemia.

Z-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol, also known as(deaminohydroxy)toremifene, having the chemical structure represented byformula, ##STR1## is a metabolite of known antiestrogen drug toremifene.Toremifene is currently used clinically for the treatment of estrogenreceptor positive breast cancer.

Z-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol has beenearlier described e.g. in Sipila, H. et al., "Metabolism of toremifenein the rat", J. Steroid Biochem., 36, 3, 211-215, (1990), in Kangas, L.,"Biochemical and pharmacological effects of toremifene metabolites",Cancer Chemother. Pharmacol., (1990), 27, 8-12, in Anttila, M. et al.,"Pharmacokinetics of toremifene", J. Steroid Biochem., 36, 3, 249-252,(1990), in Simberg, N. et al, "In vitro and in vivo binding oftoremifene and its metabolites in rat uterus", J. Steroid Biochem., 36,3, 197-202, (1990), in Bishop, J. et al., "Phase I clinical andpharmacokinetics study of high dose toremifene in postmenopausalpatients with advanced breast cancer", Cancer Chemother. Pharmacol., 30,174-178 (1992) and in the applicant's International Patent ApplicationNo. PCT/FI95/00475. It was shown that this compound is active inproducing the release of TGF-β but is devoid of significant hormonalactivity.

Now it has been found thatZ-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)-phenoxy]ethanol is active inlowering serum total and LDL cholesterol and increasing serum HDLcholesterol. Since this compound lack significant hormone associatedside effects, it is especially suitable for lowering serum total or LDLcholesterol or increasing serum HDL cholesterol, as well as in thetreatment or prevention of atherosclerosis.

The invention provides a method of lowering serum total or LDLcholesterol levels or increasing serum HDL cholesterol levels, whichmethod comprises administering to a patient in need of such treatment aneffective amount ofZ-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol.

The invention also provides a method for the prevention or treatment ofatherosclerosis, which method comprises administering to a patient inneed of such treatment an effective amount ofZ-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol.

The present invention also provides the use ofZ-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol in themanufacture of a medicament for use in lowering serum total or LDLcholesterol levels or increasing serum HDL cholesterol levels.

The invention also provides a pharmaceutical composition for use inlowering serum total or LDL cholesterol levels or increasing serum HDLcholesterol levels comprising an effective amount ofZ-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol.

The invention also provides the use ofZ-2-[4-(4chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol in themanufacture of a medicament for the prevention or treatment ofatherosclerosis.

The compound of the invention may be administered in a variety of waysincluding orally, parenterally or transdermally using conventional formsof preparations, such as capsules, tablets, granules, powders,suppositories, injections, patches, suspensions and syrups. The term"effective amount" means an amount of compound of the invention which iscapable of lowering serum total or LDL cholesterol levels or increasingserum HDL cholesterol levels. The compound of the invention may beadministered according to the method of the invention monthly, weekly ordaily or several times a day depending upon the patent's needs. Atypical daily oral dosage is within the range of from about 0.5 mg toabout 1000 mg, preferably from about 10 mg to about 800 mg, of theactive compound. However, the dosage may be properly varied depending onthe age, body weight and conditions of the patient as well as on theadministration method. The compound of the invention may be administeredalone or together with other active compounds.

The compositions according to the invention can be prepared by themethods commonly employed in the art. In addition to the active compoundthe compositions may contain pharmaceutically acceptable additivescommonly used in the art, such as carriers, binders, excipients,lubricants, suspending agents and diluents. The amount of the activecompound in the compositions of the invention is sufficient to producethe desired therapeutical effect, for example about 0.5 to 1000 mg,preferably about 10 mg to 800 mg, in unit dosage for both oral andparenteral administration.

The following examples illustrate the synthesis of the compound of theinvention.

EXAMPLES Example 1

Z-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol

(i) Z-4-[4-(2-benzyloxyethoxy)-phenyl]-3,4-diphenyl-but-3-en-1-ol

The reaction vessel was charged with toluene (790 ml), 48% aqueoussodium hydroxide (790 ml), tetrabutylammonium bromide (2.12 g, 6.6 mmol)and Z-4-(4-hydroxy-1,2-diphenyl-but-1-enyl)-phenol (50 g, 0.16 mol)prepared by the method described by U.S. Pat. No. 4,996,225. The mixturewas refluxed for 30 minutes. Benzyl-(2-bromoethyl)ether (Grobelny D. etal., Tetrahedron Letters 28, 2639-42, 1979) (41.7 g, 0.19 mol) was addedto the reaction mixture and the refluxing was continued for 2 hours.Then the mixture was cooled to room temperature, layers were separatedand aqueous phase was washed with toluene. Toluene phases were combined,washed with water, dried and evaporated to dryness. The residue was usedin the next stage without further purification.

¹ H NMR (300 MHz, CDCl₃): d 1.2 (1H, t, OH), 2.8 (2H, t, CH₂ --C═), 3.6(2H, dt, CH₂ OH), 3.7 (2H, t, CH₂ OBn), 4.0 (2H, t, CH₂ OPh), 4.6 (2H,s, OCH₂ Ph), 6.6 (2H, d, H-PhO), 6.8 (2H, d, H-PhO), 7.1-7.4 (15H, m,H-Ph).

(ii) Z-1-[4-(2-benzyloxyethoxy)-phenyl]-4-chloro-1,2-diphenyl-but-1-ene

Z-4-[4-(2-benzyloxyethoxy)-phenyl]-3,4-diphenyl-but-3-en-1-ol preparedin the previous stage was dissolved in acetonitrile (400 ml). Triphenylphosphine (103.5 g, 0.4 mol) and tetrachloromethane (120 g, 0.79 mol)were added and the mixture was refluxed for 2 hours. Then the mixturewas evaporated to dryness under reduced pressure. The residue wasdissolved in methanol (160 ml) and water (40 ml) and extracted threetimes with petroleum ether (3×200 ml) at boiling point. Petroleum etherlayers were combined and evaporated to dryness under reduced pressure.The residue was crystallized twice from ethanol (700 ml). Yield 36 g.

¹ H NMR (300 MHz, CDCl₃): d 2.9 (2H, t, CH₂ --C═), 3.4 (2H, dt, CH₂ Cl),3.7 (2H, t, CH₂ OBn), 4.0 (2H, t, CH₂ OPh), 4.6 (2H, s, OCH₂ Ph), 6.6(2H, d, H-PhO), 6.8 (2H, d, H-PhO), 7.1-7.4 (15H, m, H-Ph).

(iii) Z-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)-phenoxy]-ethanol

Z-1-[4-(2-benzyloxyethoxy)-phenyl]-4-chloro-1,2-diphenyl-but-1-ene (36g, 0.08 mol) was dissolved in the mixture of ethyl acetate (350 ml) andethanol (350 ml). Palladium on carbon (5%, 0.28 g) was added and thesolution was flushed with hydrogen gas until there was not any startingcompound left (thin layer chromatography). Palladium on carbon wasfiltered off through siliceous earth and the filtrate was evaporated.The residue was crystallized from the mixture of ethanol (155 ml) andwater (65 ml). Yield 20 g.

¹ H NMR (300 MHz, CDCl₃): d 2.9 (2H, t, CH₂ --C═), 3.4 (2H, dt, CH₂ Cl),3.84-3.89 (2H, m, CH₂ OH), 3.92-3.96 (2H, m, CH₂ OPh), 6.6 (2H, d,H-PhO), 6.8 (2H, d, H-PhO), 7.1-7.4 (10H, m, H-Ph).

The effect of Z-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanolin lowering serum cholesterol levels is demonstrated by the followingexperiments.

EXPERIMENTS

In vivo study in rats

In this study adult female Spraque-Dawley rats were used. The compoundof the invention was dissolved into vehicle (148 mM NaCl, 2.9% Magrocol3000, 0.19% Tween 20) at different concentrations (0.5-10 mg/ml). Theanimals were dosed once daily with the solutions (1 ml/kg) by oralgavage for 28 days. The control animals received mere vehicle. The finaldaily doses of the compound of the invention were 0, 0.5, 1, 5 or 10mg/kg. At the end of the treatment period blood samples were collectedby heart puncture from anesthetized animals. The separated serum sampleswere analyzed for cholesterol, cholesterol precursor molecules andselected phytosterols by gas chromatography (Miettinen T., J. Lipid.Res. 29, 43-51, 1988).

The results are shown in Table 1. The test compound decreased serumtotal cholesterol level by 34% at the highest dose level. The relativephytosterol content also decreased slightly at the highest dose level(e.g. β-sitosterol level by 20%, Table 1). There was no significantchanges in serum squalene or precursor sterol content, except in therelative lathosterol content that increased by about 50% (Table 1).

A preliminary data from a 3-month rat study supports the concept thatZ-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol is a potentserum cholesterol lowering agent: daily dose of 0.5 or 2 mg/kg decreasedserum total cholesterol level by 15-25 or 30-40%, respectively.

                  TABLE 1                                                         ______________________________________                                        Effect on serum total cholesterol, lathosterol and b-sitosterol               content in female rat after 4 weeks oral administration.                      The values (mean ± SEM) are indicated as percent of the control            values                                                                        Dose (mg/kg)                                                                            Cholesterol Lathosterol.sup.a                                                                        β-Sitosterol.sup.a                      ______________________________________                                        0.5       90.7 ± 8.4                                                                             78.3 ± 2.3                                                                            106.0 ± 7.6                               1         91.1 ± 5.0                                                                             87.2 ± 10.3                                                                           90.6 ± 6.4                                5         89.0 ± 7.5                                                                             80.1 ± 16.8                                                                           90.9 ± 6.5                                10         65.7 ± 7.0*                                                                           146.6 ± 14.74                                                                          79.6 ± 1.0*                              ______________________________________                                         .sup.a Values relative to the cholesterol content                             * = 2p < 0.05                                                            

Cholesterol biosynthesis study

The effects on cholesterol biosynthesis were also studied in vitro inHep G2 cell cultures using ¹⁴ C-acetate as cholesterol precursor. Thetest compound was added into the culture medium at concentrations from0.001 to 5 micromolar. After 2 hours the the culture was stopped and thenewly synthetized cholesterol was quantitated by thin-layerchromatography.

In the in vitro cell culture system used the test compound did notinhibit cholesterol biosynthesis directly.

Phase I clinical study

In a phase I clinical double blind study the test compound was givenperorally to healthy postmenopausal women daily for 12 weeks followed bya 2-weeks recovery period (10 subjects at each dose level). The dosesused were 25, 50, 100 and 200 mg and serum lipide values were analyzedat 2 weeks intervals. Serum total cholesterol and HDL cholesterolcontent was determined by an enzymatic PAP-method, HDL cholesterol afterPEG-precipitation. LDL-cholesterol values were calculated.

In the phase I clinical study at the dose level 200 mg the serum totalcholesterol level decreased significantly (p<0.02) from 2 weeks onwardsand the maximal decrease was about 12% from the baseline. The decreasein serum LDL level was even more pronouced (p<0.003): the decrease wasmaximally 22% from the baseline (dose level 200 mg). The test compoundtended also increase HDL-cholesterol level, the increase was significantat the 200 mg dose level (an 10% increase over baseline, p<0.04). Theserum HDL/LDL cholesterol ratio tended to increase and this increase washighly significant (p<0.0001) at the 200 mg dose level. During therecovery period the lipide values returned to the baseline level. Nosignificant changes were seen in the placebo group.

Discussion

The mechanism by which the test compound decreases serum cholesterolcontent can be either direct inhibition of cholesterol biosynthesis, aneffect on serum lipoprotein metabolism or an effect on cholesterolabsorption from the gut, or some combination of these mechanisms.According to the present data a direct biosynthesis inhibition seemsunlikely. A direct biosynthesis inhibition is not strong at thepost-squalene stages of the pathway as the accumulation of precursorsterols is minimal at the doses studied. The decrease in β-sitosterollevel may indicate that cholesterol absorption is slightly impaired.

The human data suggests thatZ-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol has beneficiallipid effects in clinical use: the cholesterol levels and especiallyharmful LDL-cholesterol level decrease markedly. In addition, anincrease in the protective HDL-cholesterol level was noticed. Insummary, the test compound is likely to inhibit or slow down the processof atherosclerosis in clinical use. The precise mechanism of action ofthe compound is to be elucidated with further experimentation.

We claim:
 1. A method of lowering serum total and LDL cholesterol levelsand increasing serum HDL cholesterol levels in a subject in need of saidcholesterol method, which comprises administering an effective amount ofZ-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol to saidsubject.
 2. A method for preventing or treating atherosclerosis in asubject in need of said treatment or prevention, which method comprisesadministering an effective amount ofZ-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol to saidsubject.
 3. A method of lowering the serum total cholesterol level in asubject in need of said lowering, which method comprises administeringand effective amount ofZ-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol to saidsubject.
 4. A method of lowering the serum LDL cholesterol level in asubject in need of said lowering, which method comprises administeringan effective amount ofZ-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol to saidsubject.
 5. A method of increasing the serum HDL cholesterol level in asubject in need of said increase, which method comprises administeringan effective amount ofZ-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol to saidsubject.
 6. A method of increasing the serum HDL/LDL cholesterol ratioin a subject in need of said increase, which method comprisesadministering an effective amount ofZ-2-[4-(4-chloro-1,2-diphenyl-but-1-enyl)phenoxy]ethanol to saidsubject.